Asthma is a chronic inflammatory disease of the airways. Anti-inflammatory drug therapy, primarily using corticosteroids, is now considered the first-line treatment in the management of all grades of asthma severity. Although corticosteroids are believed to be the most potent anti-inflammatory agents available, they do not suppress all inflammatory mediators involved in the asthmatic response. Leukotrienes, which are lipid mediators generated from the metabolism of arachidonic acid, play an important role in the pathogenesis of asthma. They produce bronchospasm, increase bronchial hyper-responsiveness, mucus production, and mucosal edema, and enhance airway smooth muscle cell proliferation and eosinophil recruitment into the airways, and their synthesis or release is unaffected by corticosteroid administration. The use of leukotriene synthesis inhibitors or leukotriene receptor antagonists as anti-inflammatory therapies in asthma has therefore been investigated. Beneficial effects of leukotriene-modifying drugs have been demonstrated in the management of all grades of asthma severity, and there is evidence that certain patient groups (such as those with exercise-induced asthma or aspirin-induced asthma) may be particularly suitable for such therapy (Salvi et al. (2001) Chest 119:1533-46).
Leukotriene B4 (LTB4), LTB4 omega-hydroxylases, and human diseases.
Leukotriene B4, or LTB4 (chemical name: 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), is a powerful inflammatory mediator derived from arachidonate by the actions of 5-lipoxygenese and leukotriene A4 hydrolase (FIG. 1) (Samuelsson et al. (1987) Science 237:1171-76). Upon stimulation, LTB4 is rapidly synthesized by inflammatory cells such as polymorphonuclear leukocytes (PMNs), macrophages, and mast cells. LTB4 has been shown to exert a wide range of biological actions, such as leukocyte activation, chemotaxis, chemokinesis, release of lysosomal enzyme, production of superoxide anion, and constriction of lung parenchyma. These effects are mainly mediated by the activation of two pharmacologically distinct cell-surface LTB4 receptors (BLTs). BLT1 is a high-affinity receptor that has been shown to be preliminarily expressed in leukocytes, whereas BLT2 is a low affinity receptor that is expressed more ubiquitously (Toda et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:575-585; Yokomizo et al. (2001) Arch. Biochem. Biophys. 385:231-241). LTB4 has been shown to play an important role in the pathogenesis of a variety of autoimmune disease, such as nephritis, arthritis, dermatitis, and obstructive pulmonary disease. The metabolism of LTB4 leading to compounds with changed capacities to activate the BLT1 and BLT2 receptors is very likely of importance for the regulation of inflammation (Kikuta et al (2002) Prostaglandins Other Lipid Mediat. 68-69:345-362). LTB4 can be structurally modified by different enzymatic pathways, i.e., by dehydrogenation of the 12-hydroxy group, by hydrogenation of the 10,11 double bond, by oxygenation of the omega-side chain, and by a combination of these reactions (Yokomizo et al. (2001) Arch. Biochem. Biophys. 385:231-241; Wheelan et al. (1999) Pharmacol. Exp. Ther. 288:326-334).
Hydroxylation of LTB4 at the omega position is considered to be specifically catalyzed by cytochrome P450 (P450 or CYP) enzymes belonging to the CYP4F subfamily (Kikuta et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:345-362; Kikuta et al. (2000) Arch. Biochem. Biophys. 383:225-232). In human PMN, LTB4 is rapidly converted into 20-hydroxy-LTB4 with a Km of about 0.6 micromolar (Powell (1984) J. Biol. Chem. 259:3082-3089). The human enzyme catalyzing this reaction has been identified as CYP4F3A, which also is referred to as human LTB4 omega-hydroxylase (Kikuta et al., J. Biol. Chem. 268, 9376-80, 1993). Binding studies have shown that 20-hydroxy-LTB4 has about the same, or even higher affinity for the BLT1 receptor than 20-hydroxy-LTB4, whereas it has an 18 times lower affinity for the BLT2 receptor (Toda et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:575-585; Wang et al. (2000) J. Biol. Chem. 275:40686-40694). The formation of 20-hydroxy-LTB4 in human PMN is considered to be the first step in a catabolic pathway (Kikuta et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:345-362; Wheelan et al. (1999) Pharmacol. Exp. Ther. 288:326-334). However, it is possible that 20-hydroxy-LTB4, due to its discrimination between BLT1 and BLT2 in combination wit its changed physical properties, could play an important direct role in inflammation (Clancy et al. (1984) Proc. Natl. Acad. Sci. USA 81:5729-33). 20-hydroxy-LTB4 can undergo another omega oxygenation step leading to the formation of 20-carboxy-LTB4, a metabolite with decreased binding affinity for both BLT1 and BLT2 (Wang et al. (2000) J. Biol. Chem. 275:40686-40694). The formation of 20-hydroxy-LTB4 has been described as being catalyzed by CYP4F3A, and by the action of alcohol dehydrogenase and aldehyde dehydrogenase (Kikuta et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:345-62; Wheelan et al. (1999) Pharmacol. Exp. Ther. 288:326-334; Baumert et al (1989) Eur. J. Biochem 182: 223-229).
LTB4 Omega-hydroxylase Genes, mRNAs, and Isoforms
Kikuta et al. (1993; J Biol Chem. 268:9376-9380) first described isolating cDNA clones for human leukotriene B4 (LTB4) omega-hydroxylase (CYP4F3) expressed in human leukocytes, encoding a protein of 520 amino acids with a molecular weight of 59,805. They determined that the amino acid sequence of CYP4F3 showed 31-44% similarity to other CYP4 family members CYP4A, CYP4B, and CYP4C, but less than 25% similarity to any of the other P-450 families.
Christmas et al. (1999; J Biol Chem 274:21191-21199) cloned a novel isoform CYP4F3 (CYP4F3B) that was expressed in fetal and adult liver, but not in PMNs. They determined that, although the CYP4F3 gene contains 14 exons and 13 introns, the cDNAs for CYP4F3A (the PMN isoform) and CYP4F3B have identical coding regions, except that they contain exons 4 and 3, respectively. Both exons code for amino acids 66-114 but share only 27% identity, and both isoforms contain a total of 520 amino acids. Moreover, the K(m) of CYP4F3B is apparently 26-fold higher than the K(m) of CYP4F3A when LTB4 omega-hydroxylase activity was measured using LTB4 as the substrate. In addition, the 5′-termini of CYP4F3A and CYP4F3B mRNAs are derived from different parts of the CYP4F3 gene, and are initiated from distinct transcription start sites located 519 and 71 base pairs (bp), respectively, from the ATG initiation codon. A consensus TATA box is located 27 bp upstream of the CYP4F3B transcription start site, and a TATA box-like sequence is located 23 bp upstream of the CYP4F3A transcription start site. CYP4F3A inactivates LTB4 by omega-hydroxylation (Km=0.68 microm) but has low activity for arachidonic acid (Km=185 microm). CYP4F3B is selectively expressed in liver and kidney, and is the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, and is able to utilize arachidonic acid as a substrate for omega-hydroxylation (Km=22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II (Christmas et al. (2001) J Biol Chem. 276:38166-38172). Thus, the tissue-specific expression of functionally distinct CYP4F3 isoforms is regulated by alternative promoter usage and mutually exclusive alternative exon splicing, result in the synthesis of two similar, but functionally distinct CYP4F3 isoforms (Christmas (2003) J Biol Chem 278:25133-25142).
Even though hydroxylation of CYP4F3A presumably occurs mainly in the PMN and in tissue infiltrated by PMNs, it is possible that metabolism in the liver also plays a role in the inactivation of LTB4. In human liver, LTB4 can be metabolized into 20-hydroxy-LTB4 by CYP4F2 and CYP4F3B. However, CYP4F2 and CYP4F3B catalyze this reaction with Km values approximately 100 times and 30 times greater, respectively, than CYP4F3A. In the liver, 20-hydroxy-LTB4 is metabolized rapidly into 20-carboxy-LTB4, which can then undergo beta-oxidation leading to 18-carboxy-dinor-LTB4. The human enzyme CYP4F3A, and possibly CYP4F2 and CYP4F3B, likely plays an important regulatory role during inflammation due to its involvement in LTB4 omega-hydroxylation (Christmas et al. (2001) J. Biol. Chem. 276:38166-38172; Kikuta et al., (1993) J. Biol. Chem. 268:9376-9380; Kikuta et al. (1994) FEBS Lett. 348:7074; Kikuta et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:345-362; Hankin et al. (1998) J. Pharmacol. Exp. Ther. 285:155-161; Wheelan et al. (1999) Pharmacol. Exp. Ther. 288:326-334; Bylund et al. (2003) Arch Biochem Biophys 412:34-41).
CYP4F3A is the most tissue specific and most efficient LTB4 omega-hydroxylase, judging from its restricted localization in human polymorphonuclear leukocytes (PMN) and its very low Km value for LTB4. In contrast, CYP4F2 is widely distributed in human liver and other tissues, and catalyzes omega-hydroxylation of various lipoxygenase-derived eicosanoids as well as LTB4, with relatively comparable and high Km values. CYP4F3B is very similar to CYP4F2 in its tissue localization and its Km value for LTB4 (Kikuta et al. (2002) Prostaglandins Other Lipid Mediat. 68-69:345-362). Until recently, it was believed that CYP4F3 was the only LTB4 omega-hydroxylase expressed in PMNs, and hence, to the degree that LTB4 contributed to asthma, it would be presumed that CYP4F3 would be most responsible for modulating its effects. However, Kikuta et al. (2004; Biochim Biophys Acta. 1683:7-15) have provided evidence that PMNs also express CYP4F3B in addition to CYP4F3A. Moreover, the transcription start site of CYP4F3B mRNA in PMNs is identical to that of CYP4F3 (i.e., CYP4F3A) mRNA (Kikuta et al. (2004) Biochim Biophys Acta. 1683(1-3):7-15). They also provided evidence that CYP4F3A is expressed at low levels in a population of peripheral blood monocytes.
There are at least four rat CYP4F enzymes, which have been designated CYP4F1 CYPF4, CYP4F5, and CYP4F6. There is significant amino acid sequence homology between mammalian CYP4F proteins. For example, the human CYP4F2, CYP4F3a, and CYP4Fb enzymes differ in amino acid sequence by 87 to 92%. Similarly, the rat CYP4F5 and CYP4F6 enzymes are 79% homologous. In addition, there is fairly substantial amino acid sequence homology between the human enzymes (CYP4F2, CYP4F3a, and CYP4Fb) and the rat enzymes (CYP4F5 and CYP4F6), which range in homology from 71% (human CYP4F2 vs rat CYP4F5) to 76% (human CYP4F3b vs rat CYP4F6). It is not surprising then, considering these amino acid sequence similarities, that each of the four rat isoforms of CYP4F (i.e., CYP4F1 CYPF4, CYP4F5, and CYP4F6), like their human counter-parts, are known to catalyze the omega-hydroxylation of LTB4 CYP4F1 and CYP4F4 also catalyze the omega-hydroxylation of arachidonic acid. Like the CYP4F3 isoforms in humans, CYP4F1 catalyzes the omega-hydroxylation of LTB4 to form 20-hydroxyl-LTB4. The rat CYP4F5 and CYP4F6 isoforms catalyze the omega-hydroxylation of LTB4 to hydroxylated forms of LTB4 not reported in humans. CYP4F5 omega-hydroxylates LTB4 to form 18-hydroxyl-LTB4, and CYP4F6 omega-hydroxylates LTB4 to form 18-hydroxyl-LTB4, and 19-18-hydroxyl-LTB4 (Bylund et al. (2003) Arch. Biochem. Biophys. 412:34-41; Chen and Hardwick (1993) Arch. Biochem. Biophys. 300:18-23; Kawashima and Strobel (1995) Biochem. Biophys. Res. Commun. 217:1137-1144; Xu et al. (2004) J. Pharmacol. Exp. Ther. 308:887-895).
The enzymatic activities of leukotriene B4 hydroxylase may be enhanced in the presence of an enzyme, NADPH-cytochrome P-450 reductase, which can convert oxidized forms of leukotriene B4 hydroxylases to reduced forms. NADPH-cytochrome P-450 reductase, which is oxidized in the process of reducing leukotriene B4 hydroxylase, can in turn be converted to a reduced form in the presence of a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). In the process of reducing NADPH-cytochrome P-450 reductase, NADPH is converted to an oxidized form (NADP+) (Sumimoto et al. (1988) Eur J Biochem. 172(2):315-324); Nisimoto et al. (1994) Biochem J. 297:585-593; Mukhtar et al. (1989) Xenobiotica 19:151-159; Kikuta Y et al. (1998) Arch Biochem Biophys 355:201-205; Bylund et al. (2003) Arch Biochem Biophys 412:34-41).